新风胶囊通过调节NLRP3通路抑制类风湿关节炎巨噬细胞焦亡

Xinfeng Capsule(新风胶囊)Inhibits Pyrophosis of Macrophages in Rheumatoid Arthritis by Regulating NLRP3 Pathway

目的观察新风胶囊含药血清对类风湿关节炎巨噬细胞极化及焦亡的影响,探讨新风胶囊治疗类风湿关节炎的可能机制。方法PMA诱导THP-1分化成巨噬细胞,INF-γ诱导巨噬细胞建立M1型巨噬细胞模型,制备XFC含药血清,用cell counting kit-8(CCK-8)、酶联免疫吸附法(ELISA)、免疫蛋白印迹(western bloting)、免疫荧光法观察XFC含药血清对巨噬细胞极化及焦亡指标的影响。结果CCK8结果显示,10 ng/m L的IFN-γ在24 h对THP-1巨噬细胞的刺激最佳,H-XFC含药血清在干预72 h后,巨噬细胞活力最佳。WB结果显示,与单纯巨噬细胞组相比,IFN-γ+巨噬细胞组iNOS、CD86、TNF-α、IL-6、IL-1β蛋白水平明显升高(P<0.01);新风胶囊含药血清干预后,iNOS、CD86、TNF-α、IL-6、IL-1β蛋白表达水平均明显降低(P<0.05或P<0.01)。ELISA结果提示,与单纯巨噬细胞组相比,IFN-γ+巨噬细胞组TNF-α、IL-6、IL-1β、IL-12表达水平明显升高(P<0.01);新风胶囊含药血清干预后,TNF-α、IL-6、IL-1β、IL-12表达水平均明显降低(P<0.01)。免疫荧光显示,与单纯巨噬细胞组相比,IFN-γ+巨噬细胞组NLRP3、Caspase1蛋白表达水平明显升高(P<0.01);新风胶囊含药血清干预后,NLRP3、Caspase1蛋白表达水平均明显降低(P<0.05或P<0.01)。结论XFC通过抑制细胞焦亡,阻止巨噬细胞向M1型巨噬细胞分化,起到治疗RA免疫炎症的作用。

Objective To observe the effect of Xinfeng Capsule(新风胶囊,XFC)-containing serum on the polarization and pyroptosis of macrophages in rheumatoid arthritis(RA)and explore the possible mechanism of XFC in the treatment of rheumatoid arthritis.Methods PMA induced the differentiation of THP-1 into macrophages.M1 type macrophage model was established by interferon-γ(INF-γ)inducing macrophages,and preparated the XFC-containing serum.Then,we observed the effects of XFC-containing serum on macrophage polarization and cell pyroptosis indexes by cell counting Kit-8(CCK-8),enzyme-linked immunosorbent assay(ELISA),western blot and immunofluorescence.Results CCK8 results showed that the optimal concentration and time of INF-γ on macrophage were 10 ng/mL and 24 h.The activity of macrophages were the best with H-XFC-containing serum treatment after 24 h.The results of western blot showed that compared with those of the macrophage group,the protein levels of inducible nitric oxide synthase(iNOS),CD86,tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-1β in IFN-γ-induced macrophage group were significantly increased(P<0.01).The expression levels of iNOS,CD86,TNF-α,IL-6 and IL-1β were significantly decreased after the intervention of XFC-containing serum(P<0.05 or P<0.01).The results of ELISA showed that compared with those of the macrophage group,the expression levels of TNF-α,IL-6,IL-1βand IL-12 in IFN-γ-induced macrophage group were significantly increased(P<0.01).The expression levels of TNF-α,IL-6,IL-1β and IL-12 were significantly decreased after intervention with XFC-containing serum(P<0.01).The results of immunofluorescence showed that compared with those of the macrophage group,the protein expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3)and Caspase1 in IFN-γ-induced macrophage group were significantly increased(P<0.01).After the intervention of XFC-containing serum,the protein expression levels of NLRP3 and Caspase1 were significantly decreased(P<0.05 or P<0.01).Conclusion XFC can inhibit the differentiation of macrophages to M1 type of macrophages by inhibiting cell pyrotosis,and plays a role in the treatment of RA immune inflammation.